Methods for obtaining the enriched fraction of ram seminal vesicle proteins (RSVP14)

ABSTRACT The objective of the present study was to develop a methodology to obtain the enriched fraction of ram seminal vesicle protein 14 (RSVP14). The study was developed using Morada Nova rams, from which semen samples were collected weekly. Seminal plasma proteins were precipitated with cold ethanol, and then 6.15 mg/mL of total proteins were subjected to liquid gelatin affinity chromatography using a Gelatin-Sepharose matrix coupled to an automated chromatographic system. Proteins were eluted into four fractions (A, B, C, and D), in which A and B contained non-gelatin-binding proteins, and C and D fractions contained gelatin-binding proteins. Gels were analyzed by Quantity One software, in which five protein bands were detected in fraction D, with molecular weights between 12 and 30 kDa. The gelatin-binding proteins (fraction D) were loaded into a HiTrap™ Heparin HP affinity column. Two chromatographic fractions were separated (D1 and D2), in which D1 contained non-heparin-binding proteins, and D2 contained heparin-binding proteins. Proteins from the last two peaks were subjected to 12.5% SDS-PAGE and Western Blot. Two bands with molecular weight of 14 and 24 kDa, contained in fraction D1, were excised from gel and subjected to tandem mass spectrometry, identifying the proteins RSVP14 and RSVP24. Thus, the chromatographic methods of the present study are efficient to capture the enriched fraction of RSVP14.

摘要 本研究旨在开发一种获取公羊精囊蛋白14（RSVP14）富集组分的方法学。本研究以莫拉达·诺瓦公羊为实验对象，每周采集其精液样本。精浆蛋白经冷乙醇沉淀后，取浓度为6.15 mg/mL的总蛋白，采用偶联于自动化色谱系统的明胶-琼脂糖（Gelatin-Sepharose）基质开展液相明胶亲和层析。蛋白被洗脱为四个组分（A、B、C、D），其中A、B组分含非明胶结合蛋白，C、D组分含明胶结合蛋白。采用Quantity One软件对凝胶进行分析，在D组分中检测到5条蛋白条带，分子量介于12~30 kDa之间。将明胶结合蛋白（D组分）上样至HiTrap™ 肝素HP亲和层析柱，分离得到两个色谱组分（D1和D2）：D1含非肝素结合蛋白，D2含肝素结合蛋白。收集最后两个峰的蛋白，进行12.5%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）与蛋白质印迹（Western Blot）分析。从凝胶中切取D1组分中分子量分别为14 kDa和24 kDa的两条蛋白条带，进行串联质谱分析，鉴定出目标蛋白为RSVP14与RSVP24。综上，本研究采用的色谱方法可高效获取RSVP14的富集组分。